Anti-Diabetic Effect of IGFBP2

8/6/2019 Anti-Diabetic Effect of IGFBP2

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Anti-Diabetic Effects of IGFBP2, a Leptin-regulated gene.

Kristina Hedbacker 1, Kivanc Birsoy 1, Robert W. Wysocki 1,2, Esra Asilmaz 1,Rexford S. Ahima 3, I. Sadaf Farooqi 4, Jeffrey M. Friedman 1,2*

1 Laboratory of Molecular Genetics, Rockefeller University, New York, NY 10065, USA

2 Howard Hughes Medical Institute, New York, NY 10065, USA

3 Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, and

Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania School of

Medicine, Philadelphia, Pennsylvania, USA.

4 University of Cambridge Metabolic Research Laboratories, Institute of Metabolic

Science, Addenbrooke's Hospital, Cambridge, CB2 0QQ, United Kingdom

*Corresponding author: Jeffrey Friedman [email protected] Phone number:

(212) 327-8000

RUNNING TITLE:

Leptin-regulated IGFBP2 corrects diabetes

SUMMARY

We tested whether leptin can ameliorate diabetes independent of weight loss by

defining the lowest dose at which leptin treatment of ob/ob mice reduces plasma

[glucose] and [insulin]. We found that a leptin dose of 12.5 ng/hour significantly lowers

blood glucose and that 25 ng/hour of leptin normalizes plasma glucose and insulin

without significantly reducing body weight, thus establishing that leptin exerts its most

potent effects on glucose metabolism. To find possible mediators of this effect, we

profiled liver mRNA using microarrays and identified IGF Binding Protein 2 as being
mailto:[email protected]:[email protected]


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regulated by leptin with a similarly high potency. Over-expression of IGFBP2 by an

adenovirus reversed diabetes in insulin resistant ob/ob, Ay/a and diet-induced obese

mice, as well as insulin deficient streptozotocin-treated mice. Hyperinsulinemic clamp

studies showed a three-fold improvement in hepatic insulin sensitivity following IGFBP2

treatment in ob/ob mice. These results show that IGFBP2 can regulate glucose

metabolism, a finding with potential implications for the pathogenesis and treatment of

diabetes.

INTRODUCTION

Leptin treatment effectively corrects hyperglycemia and hyperinsulinemia in leptin-

deficient mice and humans and other forms of diabetes (Farooqi et al., 1999; Montague

et al., 1997; Muzzin et al., 1996). However, studies of the underlying mechanism are

complicated by the fact that leptin also causes marked weight loss, which by itself can

improve diabetes. To address this, we set out to define the lowest dose of leptin that

could correct insulin resistance and diabetes and identified two low doses at which an

infusion of leptin corrected hyperglycemia without normalizing food intake or reducing

body weight. Transcription profiles from the livers of ob/ob mice treated with these

doses identified several leptin regulated genes, including IGFBP2, which weresignificantly induced even by the lowest dose of leptin (12.5 ng/hour subcutaneously), a

dose that does not significantly raise the plasma leptin level. IGFBP2 is a plasma

protein and one of 6 homologous proteins that can bind to IGFs. IGFBPs are generally

thought to inhibit the action of IGFs through high-affinity binding that prevents

interaction with IGF receptors (Dunger et al., 2004; Firth and Baxter, 2002; Kelley et al.,

1996; Kelley et al., 2002; Rosenzweig, 2004). A loss of IGF1 is known to cause

diabetic-like symptoms, as is over-expression of IGFBP1 (Crossey et al., 2000;

Rajkumar et al., 1999).

It has been suggested that IGFBP2 also inhibits IGF-1 (Firth and Baxter, 2002;

Kelley et al., 1996; Kovacs et al., 1999; Sadri and Lautt, 2000). IGF-1 is known to have

antidiabetic effects that are independent of the insulin receptor (Di Cola et al., 1997;

Froesch et al., 1996; Guler et al., 1987; Kovacs et al., 1999; Sadri and Lautt, 2000;


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Zenobi et al., 1994). If true, this predicts that over-expression of IGFBP2 should worsen

diabetes in ob/ob mice; however, this was inconsistent with the finding that IGFBP2 is

induced by a low dose of leptin that improves diabetes and raised the possibility that it

could have the opposite effect. We thus considered that acute over-expression of

IGFBP2 could improve glucose metabolism. We tested this by replicating its expression

in liver using a recombinant adenovirus, which, under the conditions of these studies,

was only expressed in liver.

Here we show that acute IGFBP2 over-expression corrected hyperinsulinemia

and hyperglycemia not only in leptin-deficient ob/ob mice but also in DIO and Ay Type 2

diabetic mice which are leptin-resistant and insulin-resistant. IGFBP2 also normalized

glucose levels of insulin-deficient mice that were treated with streptozotocin. Finally,

hyperinsulinemic euglycemic clamp studies were used to establish the physiologic

mechanism by which IGFBP2 corrects diabetes in ob/ob mice.

RESULTS

Low-dose leptin treatment of ob/ob mice corrects hyperglycemia and

hyperinsulinemia independently of body weight.

We performed a dose-response study of leptin treatment with the goal of finding aspecific low dose of leptin that does not reduce weight and food intake but that has

positive effects on diabetes. Osmotic pumps were implanted subcutaneously in ob/ob

mice with increasing doses of leptin: 0, 12.5, 25, 50, and 100 ng/hour leptin for 12 days.

Daily weight and food-intake were measured (FIG 1A, 1B). At 0, 4, 8 and 12 days, mice

were fasted for 6 hours, anesthetized and blood was drawn. (The phlebotomy

procedure resulted in a modest weight loss in all of the groups).

As expected, mice receiving the highest doses of leptin, 50 and 100 ng/hour,

showed a significant decrease in food intake and body weight with normalization of

plasma glucose and insulin. A dose of 25 ng/hour of leptin also normalized plasma

glucose and insulin despite the fact that these animals failed to lose weight during the

course of the experiment. This dose resulted in a steady state plasma [leptin] of 0.7

ng/ml which is barely above background. Finally, a leptin dose of 12.5 ng/hour, which


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fails to increase plasma leptin above background levels, significantly reduced plasma

glucose levels from 400 ng/mL to 208 ng/mL, p


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p


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effects of the viral infection. As a control, we used additional adenovirus strains with no

insertion or with an insertion of a luciferase reporter. The luciferase virus allowed us to

assess the sites of gene expression from the viral vector. Five days after intravenous

injections of the Ad-luciferase adenovirus, mice received an intraperitoneal injection of

luciferin and were imaged using a CCD camera (IVUS, Caliper Technology). These data

showed that viral gene expression was limited to the liver, the site of endogenous

IGFBP2 expression, and also at the site of injection in the tail (Supplemental Figure 2A).

Animals injected with the IGFBP2 adenovirus showed a highly significant

increase in plasma IGFBP2 levels to at least 4000 ng/mL (data not shown). The

IGFBP2 and empty (control) adenoviruses were injected into ob/ob mice followed by

measures of daily body weight and food intake (FIG 3A and 3B) and plasma glucose

and insulin five days after viral injection (FIG 3C and 3D). Mice treated with the IGFBP2

adenovirus showed a modest decrease in food intake with a stabilization of body weight

while the control ob/ob mice continued to gain weight (FIG 3A and 3B). At 5 days post-

injection, the ob/ob mice that had received the IGFBP2 treatment had completely

normalized plasma glucose and insulin. While control mice had blood glucose levels of

over 300 mg/dL, IGFBP2 treated animals had blood glucose levels under 100 mg/dL

(320 vs. 94 mg/dL for the controls, p


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animals compared to controls (36 mg/kg/min in IGFBP2-treated ob/ob vs. 10 mg/kg/min

in controls, p


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5G and 5H). Changes in food intake and body weight were minimal and in most cases

not statistically significant (FIG 5C and 5D).

Ad-IGFBP2 treatment corrects hyperglycemia in insulin deficient mice.

Previous studies have shown that leptin treatment corrects hyperglycemia in Type 1

diabetic mice (Yu et al., 2008). We next tested whether IGFBP2 can improve diabetes in

this setting of insulin deficiency. IGFBP2 was injected into streptozotocin-induced Type

1 diabetic/insulin deficient mice. Plasma insulin was not detectable 6 weeks post low-

dose STZ-treatment even when using an ultra-sensitive mouse insulin EIA kit (FIG 5B

and data not shown). At day 5 after IGFBP2-injections, control mice had fasting (4 hour)

glucose levels of 509 as compared to 136 mg/dL of the IGFBP2 treated group, p


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Leptin has been shown to improve hyperglycemia and insulin resistance in a number of

clinical settings in animals and humans. These conditions include leptin

mutations/deficiency, lipodystropy, Rabson-Mendenhall syndrome, resulting from insulin

receptor mutations, and more recently Type 1 diabetes in NOD mice, which have a

complete insulin deficiency (Asilmaz et al., 2004; Farooqi and O'Rahilly, 2004;

Montague et al., 1997; Oral et al., 2002; Petersen et al., 2002; Yu et al., 2008). The

mechanism by which leptin exerts these salutary effects is poorly understood. In this

report, we sought to explore the mechanism responsible for leptins anti-diabetic effects

and first showed that leptin can correct diabetes of ob/ob mice at low doses that do not

significantly reduce body weight. IGFBP2 gene expression is induced by these low

doses of leptin treatment and IGFBP2 plasma levels increase in response to leptin

treatment of ob/ob and wild type mice. Over-expression of IGFBP2 using a recombinant

adenovirus resulted in a striking reduction of plasma glucose and insulin in all animals

tested including ob/ob, wild type mice, as well as Ay, DIO, and streptozotocin treated

animals. Overall, these data confirm that leptin can improve glucose homeostasis

independent of its ability to reduce weight and suggest that IGFBP2 may account for a

portion of leptins anti-diabetic effects.

IGFBP2 is a 34 kD plasma protein produced by liver. It was originally isolated

based on its ability to bind to IGF1 and IGF2 and is one of six IGF binding proteins thatcirculate in plasma (Baxter and Martin, 1989; Kelley et al., 2002; Martin and Baxter,

1986). Further studies in vitro suggested that IGFBP2 and the other IGFBPs function as

IGF inhibitors by chelating these ligands (Hoflich et al., 1998; Jones and Clemmons,

1995). However, IGFBP2 circulates at equimolar, or lower molar, concentrations

compared to IGF1 which is not a typical characteristic of hormone inhibitors that

generally circulate in molar excess of the ligand. This raises the possibility that IGFBP2

may not inhibit IGF-1 in vivo or might have IGF-1 independent effects. In vivo, IGFBP2

has been invoked as playing a role to modulate IGF signaling in growth and

development, cancer and diabetes. The precise function of IGFBP2 is poorly

understood and it is not well established whether it inhibits or activates IGF1 signaling

in vivo or if it has actions independent of IGF (Wolf et al., 2000). (These possibilities are

not mutually exclusive).


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An IGFBP2 knockout does not have a major metabolic phenotype and it as been

suggested that other IGFBPs can compensate for its loss (Pintar et al., 1995; Wood et

al., 1993). However, a possible role for IGFBP2 in the regulation of metabolism has

been suggested by its association to diabetes in several human whole genome studies

(Cauchi et al., 2008a; Cauchi et al., 2008b; Grarup et al., 2007; Hertel et al., 2008;

Horikawa et al., 2008; Sanghera et al., 2008; Saxena et al., 2007; Scott et al., 2007;

Zeggini et al., 2007). In addition, a CMV-IGFBP2 transgene has been shown to

modestly prevent weight gain and hyperglycemia in DIO mice. However, in that report, it

was not shown whether the effect of IGFBP2 on diabetes was independent of its effect

on body weight nor was the potential therapeutic benefit of acute IGFBP2 over-

expression established (Wheatcroft et al., 2007).

The results reported here show an ability of IGFBP2 to profoundly reduce plasma

glucose and insulin when acutely over-expressed from an adenoviral vector in wild type

and ob/ob mice as well as markedly hyperglycemic STZ-induced Type 1 diabetic mice

and even in leptin resistant DIO and Ay mice, both of which have Type 2 diabetes (Lin

et al., 2000; Prpic et al., 2003; Van Heek et al., 1997). Thus IGFBP2 can improve

glucose metabolism in leptin sensitive and leptin resistant animals, an observation that

is consistent with the finding that IGFBP2 is regulated by, and thus downstream of,

leptin signaling. IGFBP2 levels are not higher in DIO and Ay animals than in wild typemice, which is consistent with the fact that they are leptin resistant. The observation that

these leptin resistant animals still respond to exogenous IGFBP2 similarly to leptin

sensitive animals is also consistent with IGFBP2 being downstream of leptin signaling in

brain.

In these studies, we used an adenovirus to over-express IGFBP2 because this

protein has 7 disulphides and is difficult to produce as a recombinant protein in sufficient

amounts to perform the studies reported here. Adenoviral expression results in IGFBP2

protein levels that are significantly higher than the level at which it normally circulates in

the blood stream and further studies will be required to determine whether the observed

effects are physiologic. Efforts to produce bioactive recombinant IGFBP2 are underway.

The mechanism by which IGFBP2 reduces blood glucose is also under

investigation. Hyperinsulinemic euglycemic clamp show that IGFBP2 markedly


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improves hepatic insulin-sensitivity in ob/ob mice with a three-fold increase in the

glucose infusion rate. However, it is important to point out that despite the infusion of

more than 10 times the insulin dose typically given to wild type mice, the glucose

infusion rate was still lower, and the HGP in IGFBP2-treated ob/ob mice was still higher,

than the levels seen in wild type mice (Ahima et al., 2006; Qi et al., 2006). We propose

that the improvement in hepatic insulin sensitivity in IGFBP2-treated ob/ob mice is due,

at least in part, to a reduction in hepatic steatosis. A similar negative correlation

between hepatic steatosis and insulin sensitivity has been described in ob/ob mice

treated with an insulin-sensitizing aminosterol (Takahashi et al., 2004) or antisense

oligonucleotides against the lipid droplet protein ADRP (Imai et al., 2007). Further

studies will be required to determine to what extent the amelioration of steatosis can

account for the beneficial effects of IGFBP2 on hepatic glucose production. Further

studies will also be necessary to determine whether IGFPB2 improves steatosis and

hepatic insulin sensitivity by an autocrine mechanism or indirectly via modulating the

activity of other organs or the levels of other hormones.

Since IGFBP2 does not fully correct HGP but nonetheless normalizes plasma

glucose, insulin and a GTT in ob/ob mice, it is possible that it could also have additional

insulin independent effects to reduce blood glucose. This possibility is consistent with

the observation that the IGFBP2 adenovirus can also normalize glucose levels in insulindeficient STZ mice. While it is conceivable that IGFBP2 can sensitize these mice to a

low level of residual insulin, it is also possible that IGFBP2 acts through an insulin or

even IGF independent mechanism. Studies to further explore the hepatocellular

mechanism by which IGFBP2 reduces blood glucose are currently underway.

The data in this report also establish that the potency of leptin for reducing

hyperglycemia is greater than that for correcting food intake and body weight. While an

early study of leptin showed direct effects on hyperglycemia independent of weight loss

in ob/ob mice, the study used daily IP bolus injections of leptin as opposed to the

continuous doses used in this study (Pelleymounter et al., 1995). In other studies, leptin

improved diabetes to a greater extent than pair-feeding, suggesting that leptin had

independent effects on glucose metabolism (Levin et al., 1996; Schwartz et al., 1996).

However, these studies did not identify the mechanisms by which leptin improved


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diabetes without reducing weight. Finally a study of low dose leptin infusions to treat

diabetic lipodystrophic animals was limited by the fact that the underlying condition of

these animals made it difficult to assess whether there was significant weight loss

(Asilmaz et al., 2004). Thus our data confirm that leptin regulates glucose metabolism

independent of its effects on energy balance and identify conditions under which this

effect can be studied without a confounding effect on body weight.

The low doses of leptin used in these studies also reveal a potent effect of leptin

to regulate several liver genes, some of which could also play a role in mediating

leptins effects. Previous studies of a brain and liver specific knockout of the leptin

receptor, and a brain specific leptin receptor transgenic mouse, have indicated that the

brain is the primary site of leptins actions and that its effects on liver are indirect (Cohen

et al., 2001). The nature of the signal responsible for the induction of IGFBP2 by leptin

is unknown but does not appear to be insulin, as acute increases or subacute

decreases of plasma [insulin] do not alter circulating IGFBP2 levels (Supplementary

figure 4 and Figure 2C). Further studies are necessary to establish whether leptin

regulation of IGFBP2 is mediated by efferent neural outputs from the CNS and/or a

result of modulation of its gene expression by other hormones.

Finally, consistent with the mouse data, we found lower IGFBP2 levels in leptin-

deficient human subjects compared to control subjects, and that IGFBP2 levelsincreased after leptin treatment in two out of three patients. This raises the possiblity

that IGFBP2 could have therapeutic effects in human diabetes, which would require the

use of recombinant protein rather than a viral vector. A variety of eukaryotic expression

systems will be tested for their ability to yield bioactive IGFBP2.

In summary, we have developed a protocol in which leptin treatment potently

improves diabetes independent of its ability to correct weight and food intake. This

protocol was used to identify IGFBP2 as a leptin regulated gene whose expression is

correlated with leptins anti-diabetic effect. IGFBP2 over-expression reduces blood

glucose in wild type and diabetic mice and potently suppresses heptic glucose

production suggesting that it may play a role in mediating some portion of leptins anti-

diabetic effects. Further studies will reveal whether IGFBP2 shows similar anti-diabetic

effects in clinical settings.


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EXPERIMENTAL PROCEDURES

Mice and Diet

Leptin dose experiment: 8 week old male C57Bl6 Lep-ob/ob mice were obtained

from the Jackson laboratory (Bar Harbor, ME), acclimated to our mouse facility under

controlled light conditions (12 hr light/12 hr dark), temperature (22C), single-caged and

fed ad libitum a standard mouse chow for at least a week. Alzet 2002 mini-osmotic

pumps (Alzet, Palo Alto, CA) were filled with indicated concentrations of leptin (Amgen,

Thousand Oaks, CA) and incubated at 37C in sterile 0.9% NaCl overnight and

implanted subcutaneously in 9-11 weeks old mice. Mice were single-caged and their

weight and food-intake were recorded daily. At 0, 4, 8, and 12 days of treatment, mice

were anesthetized and blood was collected intraorbitally. At day 12, mice were

anesthetized, livers were collected fresh frozen in liquid N2 or fixed in Accustain

Formalin Solution Neutral Buffered 10% Formalin (Sigma-Aldrich, St Louis, MO)

overnight for histology.

Ad-IGFBP2 experiments: 8 week old male C57Bl6 Lep-ob/ob or wild type mice

were acclimated as above. Ay mice were ordered from Jackson laboratory and kept

until at least 12 weeks of age. Wildtype male mice were ordered from Jackson

laboratories and kept on a standard high-fat-diet chow until at least 15 weeks of age.Mice received intrajugular vein injections under anesthesia (isoflurane) of 1.2x10^11

particles of Ad-CMV-empty, Ad-CMV-Luciferase or Ad-CMV-IGFBP2 (ViraQuest Inc.). 1

week after injections, plasma levels of IGFBP2 were confirmed with an IGFBP2 EIA kit

(Alpco).

Streptozotocin-treated mice: 5 week old male C57Bl6 wild type mice were

obtained from the Jackson laboratory (Bar Harbor, ME) and received a daily

intraperitoneal dose of 50 mg/kg Streptozotocin (Sigma) for 5 days. 4 weeks later,

hyperglycemic mice were injected with Ad-IGFBP2 as described above.

Metabolic studies

Glucose tolerance test: Mice were injected intraperitoneally with 1 unit

glucose/gram body weight. Blood glucose was recorded at 0, 30, 60, and 120 minutes


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post-injection. In STZ mice, blood glucose was recorded at 0 and 45 minutes post-

injection.

Hyperinsulinemic euglycemic clamp: This has been previously described in (Qi et

al., 2006). 10 week old male ob/ob mice were treated with Ad-CMV-IGFBP2 or Ad-

CMV-control via a tail vein injection, and insulin clamp was performed 10 days later. An

indwelling catheter was inserted in the right internal jugular vein under sodium

pentobarbital anesthesia and extended to the right atrium. After regaining their

presurgery weight (4 days), the mice were fasted for 6 hours, a bolus injection of 5 Ci

of [3-3H] glucose was administered, followed by continuous intravenous infusion at 0.05

Ci/min. Baseline glucose kinetics was measured for 60 min. A priming dose of regular

insulin (40 mU/kg, Humulin; Eli Lilly, Indianapolis, IN) was given intravenously, followed

by continuous infusion at 30 mUkg-1min-1. Blood glucose was maintained at 120-140

mg/dL via a variable infusion rate of 30% glucose. At the end of the 120-minute clamp,

10 Ci 2-deoxy-D-[1-14C]glucose was injected to estimate glucose uptake. The mice

were euthanized, and liver, perigonadal fat (WAT), and soleus/gastrocnemius muscle

were excised, frozen immediately in liquid nitrogen, and stored at 80C for subsequent

analysis of glucose uptake. The rates of basal glucose turnover and whole body glucose

uptake are measured as the ratio of [3H] glucose infusion rate (dpm) to the specific

activity of plasma glucose. Hepatic glucose production (HGP) during clamp is measuredby subtracting the glucose infusion rate (GIR) from the whole body glucose uptake (Rd).

Liver triglycerides: Liver triglycerides were determined using Sigma TR0100

Triglyceride Determination Kit.

Human subjects

Fasting plasma samples were obtained from three children with leptin deficiency before,

and one and six months after treatment with recombinant human leptin as reported

previously (Farooqi et al., 2002). All samples were stored at minus 80C and thawed

once prior to analysis. Results were compared to age and BMI matched controls on

whom fasting plasma samples had been obtained.

RT-PCR and microarray


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Total RNA was isolated by homogenizing liver tissue in TRIzol reagent (Invitrogen) and

purifying the RNA using Qiagen RNA prep kit (Qiagen). Real-time PCR was performed

using the TaqMan system (Applied Biosystems) according to the manufacturers

protocol as previously described (Birsoy et al., 2008). Microarrays were done using

MouseRef-8 v2 BeadChip (part# 11288185) after labeling the RNA with Ambions

Illumina TotalPrep RNA Amplification Kit.

Serum Assays

Blood glucose was determined using an Ascensia Elite XL glucometer (Bayer) or

QuantiChrom Glucose Assay Kit (BioAssay Systems, Hayward, CA). For all other

assays, mice were bled intraorbitally while anesthetized with isoflourane. Blood was

spun for 10 minutes and plasma collected. Plasma insulin was determined using an

Insulin (mouse) EIA kit (Alpco Diagnostics, Windham, NH). Plasma leptin and IGFBP2

levels were determined using a Leptin (mouse/rat) EIA kit and an IGFBP-2 (mouse/rat)

EIA kit, respectively (Alpco Diagnostics, Windham, NH). Plasma IGF 1 was determined

using a Mouse IGF-I Quantikine ELISA Kit (R&D Systems).

Histology

Paraffin-embedded, 10% formalin-perfused livers were sectioned and stained withHematoxylin and Eosin.

Statistical Analysis

All data was analyzed for statistical significance using the students t-test. P-values as

indicated.

ACKNOWLEDGMENTS

We would like to thank Roger Unger, Domenico Accili, Allyn Mark, Stephen ORahilly

and Paul Cohen, for constructive criticism on experiments and this manuscript. We

would also like to thank Shaheen Kabir, and Susan Korres for technical assistance.

Lastly, we would like to thank Ravindra Dhir at the University of Pennsylvania Diabetes


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Endocrinology Research Center (DERC) Mouse Metabolic Phenotyping Core for

performing the clamp and radioisotopic tracer studies. The DERC is supported by NIH

grant P30-DK-19525.


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FIGURE LEGENDS

Figure 1

Low-dose leptin treatment of ob/ob mice corrects blood glucose and

hyperinsulinemia independently of body weight. Mice receiving 12 day leptin

treatment. Dose of leptin as indicated in 1A. Mice fasted for 6 hours prior to receiving

anesthesia and blood collection at day 0, 4, 8, and 12. For each group, n4. A) Percent

change in body weight during 12 day leptin treatment. Arrows show day 4 and 8 of

treatment. Dotted line indicates food restricted animals (see figure 3A, 3B. 3C and 3D).

B) Food intake in grams each day during treatment. Dotted line indicates food restricted

animals (see figure 3A, 3B. 3C and 3D). C-E) ng/mL plasma leptin, mg/dL blood

glucose, and ng/mL plasma insulin at day 12 of leptin treatment. Blood glucose and

plasma insulin for food-restricted animals can be found in Figure 3C and 3D. Error bars

show standard error. * p


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food intake in grams. Food intake is average for 24 hours. Mice injected with adenovirus

on day 0. Arrow indicates 18-hour fast (for GTT). X-axis indicates day of experiment.

Dotted line shows body weight and food intake of mice pair-fed to the IGFBP2 treated

mice. C) and D) plasma glucose and plasma insulin in treated, control, and pair-fed

mice. E) Milligrams triglycerides per gram liver tissue in ob/ob control, ob/ob+IGFBP2,

ob/ob+12 days 100 ng.hr leptin and ob/ob+ 12 days of 25 ng/hr leptin. F) H&E stained

liver paraffin sections of treated and control mice. 10x and 40x as indicated. * p


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A) Serum IGFBP2 in leptin deficient and age and weight-matched controls. B) Serum

IGFBP2 in 3 leptin deficient patients before (light grey), and 6 months after (dark grey)

low-dose leptin treatment.


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Anti-Diabetic Effect of IGFBP2

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